Methods: Serum was collected from seven
BD patients with complicated cataract, and from nine controls with
uncomplicated cataract, before cataract surgery, and again 1, 7, 30,
and 90 days after surgery. In addition, aqueous humor was collected at
commencement of surgery. The protein levels of IL-23, IL-27, IL-17, and
IFN-γ in the serum and in the aqueous humor were measured by an
enzyme-linked immunosorbent assay. A laser flare-cell photometer was
used to quantify intraocular inflammation.

Results: Serum IL-23, IL-27, and IFN-γ
levels were significantly increased after cataract surgery in the BD
versus the control patients. In the BD patients, serum levels of IFN-γ
and IL-27 correlated strongly with aqueous flare values and cell
counts. Remarkably, the levels of serum IL-27 were significantly
associated with serum IFN-γ levels in BD patients (r=0.796; p=0.002).

Conclusions: Our data indicates that
serum IFN-γ and IL-27 levels are significantly elevated in BD versus
control patients and are strongly associated with post-operative
intraocular inflammation.

Behcet disease (BD) is a chronic multisystem disorder characterized
by recurrent uveitis, oral aphthae, genital ulcers, and skin lesions.
Behcet uveitis is one of the most common types of uveitis occurring in
China [1].
Cataract formation, the most frequent anterior segment complication of
uveitis in BD patients, is a major cause of decreased visual acuity [2]. Treatment of these
cataracts is not easy because surgical procedures can provoke
inflammation. Suppression of the inflammatory response is critical for
the success of surgery in these patients.

Although the pathogenesis of BD is still unclear, several reports
suggest that an autoimmune response may play an important role in the
development of inflammation in these patients [3]. Previous studies
have suggested that interferon-γ (IFN-γ) are present in BD patients [4,5]. Recent studies have
shown increased levels of other T-helper type 17 (Th17) cell associated
cytokines, such as interleukin 23 (IL-23) and IL-17, in BD patients
with active uveitis [6].
In
addition, previous studies have shown that the presence of IL-27 may
limit Th17 mediated uveitis [7].
However,
the association of these cytokines with postoperative
intraocular inflammatory activity in BD patients is not yet clear. In
this study, we determine the correlation between the serum
concentrations of these cytokines in BD patients and the intraocular
variables of the disease activity.

Patients

Seven eyes with complicated cataracts, from seven BD patients, were
included in the study. BD disease was diagnosed according to the
criteria determined by the International Study Group for BD disease [1,8]. Cataract surgery
was performed on these patients after at least 3 months of inactive
uveitis. These patients had received prednisone at a low dose
(<20 mg/d), but no other immunosuppressive agents, for at least
2 months before the first sampling. Nine eyes of nine uncomplicated
cataract patients (age related cataract patients, n=6; congenital
cataract patients, n=3) were selected as controls, were matched for age
and sex and the surgery was performed on the same day as the BD
patients. None of these controls had suffered previous ophthalmic
disease or received any medication known to influence cataract
formation. Patients and controls underwent phacoemulsification between
July 2008 and March 2009 at Zhongshan Ophthalmic Center (Guangzhou,
China). This study was performed in accordance with the Declaration of
Helsinki and with the approval of the local ethical committee. Informed
consent was obtained from all patients and controls.

Post-operative management

Standard cataract procedure was performed by the same experienced
cataract surgeon (Yizhi Liu) on all patients as described [9,10]. All patients
received dexamethasone-tobramycin eye drops four times daily during the
first week, and twice a day during the second week, after which
treatment was discontinued except when signs of severe postoperative
inflammation were present. Patients with BD disease also received
prednisolone (initial dose, 1 mg/kg), which was gradually reduced,
based on the extent of intraocular inflammation.

Measurement of anterior intraocular inflammation

Aqueous flare measurements and cell counts were made using a slit
lamp and laser flare-cell photometer FC-2000 (Kowa, Tokyo, Japan) as
described in previous studies [9-11]. Measurements of
flare intensity and cell count were done before surgery with follow-up
measurements 1, 7, 30, and 90 days after surgery. Three individual
measurements from each eye were averaged; measurements affected by
artifacts were discarded. Flare and cell readings were expressed as
photon counts per millisecond, and cells per 0.5 mm3,
respectively.

Sample collection

Blood samples (2 ml) were collected before surgery and 1, 7,
30, and 90 days after surgery. Samples of aqueous humor (AqH;
50–100 µl) were collected from the paracentesis site for
phacoemulsification. After centrifugation at 300× g for 5 min,
serum and AqH samples were separated into cellular components and
supernatant and were frozen at −80 °C until use.

Enzyme-linked immunosorbent assay

The concentrations of IL-23, IL-27, IL-17, and IFN-γ in sera or AqH
were measured with commercial sandwich enzyme-linked immunosorbent
assays, following the manufacturer’s instructions (DuoSet ELISA
Development System; R&D Systems, Minneapolis, MN). The limit of
detection was 15 pg/ml.

Statistical analysis

The data were analyzed using SPSS 10.0 for Windows XP (SPSS Science,
Chicago, IL). All data were expressed as mean±SD. Variables at the
follow-up times were compared using Student’s t test. Linear
regression was used to assess the influence of potential risk factors
(age, sex, “phaco-time” and “phaco-energy”) on flare values and cell
counts. Correlation coefficients were calculated with the Spearman
correlation coefficient test. A p≤0.05 was considered statistically
significant.

General data of the BD patients and control subjects

Patient demographics and clinical features are summarized in Table 1.
Patient ages ranged from 15 to 52 years in the BD disease group and
from 17 to 60 years in the control group. Preoperative and
postoperative courses were uneventful in all patients. Other variables
(such as age, sex, cataract type, phaco-time, phaco-energy and
phaco-time × phaco-energy) were not correlated with flare
values, cell counts, and cytokine levels in the serum and the AqH.

Cytokine levels of BD patients and controls before surgery

Before surgery, IL-17 was undetectable in the serum of all BD
patients and control subjects. IL-23 was expressed in serum of both BD
patients and control subjects. The low concentrations of IL-27 and
IFN-γ were detected in serum of BD patients. However, IL-27 and IFN-γ
were lower than the detection level in control subjects (Figure 1).
AqH samples were collected at the initiation of cataract surgery, and
protein levels of the Th17-associated cytokines were measured. The
results showed that IL-23, IL-27, IL-17, and IFN-γ were undetectable in
all AqH samples of both BD patients and controls.

The change in cytokine levels of BD patients following surgery

In BD patients, serum IL-23, IFN-γ, and IL-27 levels significantly
increased and peaked on day 1 post-surgery. Then IFN-γ and IL-27
levels decreased rapidly during the first week, although IL-23 level
still elevated. All these cytokines gradually returned to preoperative
levels by 90 days after surgery (Figure 2). IL-17 was undetectable in
serum of BD patients after surgery.

The change in cytokine levels of controls following surgery

In control subjects, serum IL-23 levels increased on day 1
post-surgery, decreased during the first month, and gently elevated on
day 90 post-surgery. IL-17, IFN-γ, and IL-27 were still lower than the
detection level after surgery (Figure 3).

Comparison of cytokine levels between BD patients and controls

Serum IL-23 levels were considerably higher in BD patients compared
to control subjects preoperatively and on days 1, 7, 30, and 90 days
postoperatively (p=0.003, p<0.001, p<0.001, p<0.001, and
p=0.007, respectively; Figure 4).

The results showed that, for both groups, the aqueous flare value
and cell count increased after surgery and achieved the highest level
on day 1 post-surgery, and then decreased rapidly during the
first week, which was then followed by a more gradual reduction. Serum
IFN-γ and IL-27 levels correlated strongly with aqueous flare value and
cell counts in BD patients, but that serum IL-23 levels did not (Table 2
and Figure 5).
Of
note, serum IL-27 levels in BD patients were significantly
associated with serum IFN-γ levels (r=0.796; p=0.002). No correlation
has been found between serum IL-23 levels and postoperative
inflammation, although serum IL-23 levels were elevated after surgery
in control subjects.

Cataract, resulting from chronic intraocular inflammation or from
the corticosteroid therapy given for inflammation, is a common
complication of uveitis in BD patients. In both children and adults,
the importance of preoperative and postoperative inflammation control
is paramount [12,13]. The Th17 cell is
a unique CD4-positive (CD4+)T-cell subset characterized by the
production of IL-17. Recent data from humans and mice suggests that
Th17 cells play an important role in the pathogenesis of a diverse
group of immune-mediated diseases, including BD and
Vogt-Koyanagi-Harada (VKH) disease [6,9,14,15]. It has been shown
that BD patients with active uveitis exhibit higher IL-17 and IL-23
levels as compared with patients with inactive uveitis, or normal
controls [6].

IL-23, a member of the IL-12 family of cytokines, plays an important
role in Th17 maintenance [16].
In
the present study, we measured the dynamic changes of serum IL-23 in
BD patients and control subjects both before and after surgery. The
results showed that serum IL-23 was upregulated in BD patients,
compared with controls, both before and after surgery. However, no
statistical association was found between the increased serum IL-23
levels and intraocular inflammation in BD patients. The exact role of
IL-23 on postoperative uveitis is not well understood and further
studies are needed to define this in more detail.

Previous studies found that an extremely Th1-rich cytokine
environment was formed in Behçet uveitis [4,17].
The
present study was also done to evaluate the influence of cataract
surgery on IFN-γ expression. Our results showed significantly
upregulated serum levels of IFN-γ after surgery in BD patients which
correlated with inflammatory variables of postoperative uveitis. These
results are consistent with those of previous studies that documented
that BD patients with active uveitis showed increased levels of IFN-γ
in both the serum and the AqH as compared to normal controls [4-6,14]. Unexpectedly, it
has been shown that almost half of all the Th17 cells co-express IFN-γ
in humans, and that IFN-γ may inhibit IL-17 expression [6,16,18]. The exact
relationship between IL-17 and IFN-γ in postoperative uveitis is not
well understood and further studies are needed to describe this in more
detail.

The immunologic role of IL-27 has been documented and discussed in
relation to various Th1/Th17-mediated inflammatory diseases [19,20]. However, its
importance in postoperative uveitis is unknown. In this study, we
invested the variation in the levels of serum IL-27 before and after
surgery. The results showed that serum IL-27 levels in BD patients were
significantly elevated after surgery and correlated significantly with
disease severity and serum IFN-γ levels. These findings are consistent
with earlier reports about the cytokine environment in psoriasis [21]. In addition, it
has been shown that IL-27, as constitutively expressed in retinal
ganglion and photoreceptor cells, is upregulated by IFN-γ, and
inhibited Th17 proliferation in uveitis [7]. Thus, the results of our study indicate that
elevated IL-27 in the sera of BD patents after surgery plays an
important role in postoperative inflammation. Further studies are
needed to assess whether IL-27 plays a protective or pathogenic role in
postoperative uveitis in BD patients.

Although IL-17 was undetectable in the serum of all subjects in the
present study, a previous study found that IL-17 production by CD4+
T cells increased significantly in the presence of IL-23 in BD patients
with active uveitis [6].
In
addition, it was reported that IL-17 is present in active uveitic
eyes in BD patients [4].
However,
in our study, IL-23, IL-17, IL-27, and IFN-γ were undetectable
in the AqH of both BD patients and controls. These results might be
attributed to the inactive stages of inflammation in BD patients, to
the use of topical corticosteroids and to the different assays used for
study.

Our study showed that the serum levels of IL-23, IFN-γ, and IL-27
were significantly elevated after cataract surgery in BD patients.
These findings support previous reports about the effects of cataract
surgery on age-related cataract patients and patients with VKH disease [9,22]. A significant
increase of serum levels of cytokines in cataract patients following
cataract surgery, have been described in those studies. In addition,
serum cytokines relating to the activity of the inflammatory response
in BD patients were most prominent in several studies [23,24]. As memory T cells
can be potent inducers of the cytokines tested, the increased serum
levels of IL-23, IFN-γ, and IL-27 following surgery strongly suggest an
underlying inflammation and immune hypersensitivity reaction in
patients with BD disease that is provoked by the cataract trauma. These
findings may be associated with several factors: a more severe
postoperative disturbance of the blood-aqueous barrier, a greater uveal
reaction to surgical trauma and some systemic response triggered in the
circulation. Further studies are needed to assess the real role of
these cytokines in postoperative uveitis in BD patients.

In conclusion, this study showed that increased serum levels of
Th17-related cytokines, particularly of IL-27 and IFN-γ, are possibly
involved in the development of postoperative inflammation in BD
patients. However, given the meticulous cross-regulatory mechanisms in
Th17 cytokines, further study is needed to investigate their detailed
role in the postoperative ocular inflammation of different aspects of
uveitic diseases.